STUDY OF FORMULATION VARIABLES ON BIOAVAILABILITY …

Chaudhari et al. European Journal of Pharmaceutical and Medical Research

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STUDY OF FORMULATION VARIABLES ON BIOAVAILABILITY OF METFORMIN

HYDROCHLORIDE

S. P. Chaudhari*, Vijaya Dhumal, S. C. Daswadkar and D. S. Shirode

Dr. D.Y. Patil College of Pharmacy, Akurdi, Pune-44.

Article Received on 09/09/2016 Article Revised on 29/09/2016 Article Accepted on 19/10/2016

INTRODUCTION

Oral route is most popular for systemic effect due to its

easy of ingestion, pain, avoidance, versatility and most importantly, patient compliance. Immediate Release

Tablets are those tablets which are designed to

disintegrate and release their medication with no special

rate controlling features, such as special coatings and

other techniques.[1] The main aim in the process of drug

development is to obtain a drug product with a good oral

bioavailability.

Diabetes is a chronic disease that occurs either when the

pancreas does not produce enough insulin or when the

body cannot effectively use the insulin it produces. Insulin is a hormone that regulates blood sugar.

Hyperglycemia, or raised blood sugar, is a common

effect of uncontrolled diabetes and over time leads to

serious damage to many of the body's systems, especially

the nerves and blood vessels.[2]

The number of people with diabetes has risen from 108

million in 1980 to 422 million in 2014. The global

prevalence of diabetes among adults over 18 years of age

has risen from 4.7% in 1980 to 8.5% in 2014. Diabetes

prevalence has been rising more rapidly in middle- and

low-income countries. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and lower

limb amputation.

In 2014, 8.5% of adults aged 18 years and older had

diabetes. In 2012 diabetes was the direct cause of 1.5

million deaths and high blood glucose was the cause of another 2.2 million deaths.[3]

Metformin hydrochloride (HCl) is a first line drug for

Diabetes Mellitus but having low bioavailability. It is

BCS class III drug, having high solubility and low

permeability through intestinal mucosa, so it is necessary

to increase the permeability of the drug and thus

bioavailability. Metformin HCl to enhance its absorption

by oral ingestion is the most convenient and commonly

used route of drug delivery. Metformin HCl acts by the

initial activation of AMPK a liver enzyme that plays an important role in insulin signaling, whole body energy

balance and the metabolism of glucose and fats. This

causes the increased peripheral utilization of glucose

may be due to improved insulin binding to insulin

receptors. Metformin HCl administration also increases

AMPK activity in skeletal muscle. AMPK is also known

to cause Glucose transporter type 4(GLUT4)

deployments to the plasma membrane, resulting in

insulin-independent glucose uptake.[4]

Other antidiabetic agents lack efficacy and also have

undesirable side effects. For instance, Insulin secretagogues result in weight gain, hypoglycemia and

inability to protect 𝛽 cells from death.

Thiazolidinediones result in weight gain and kidney

SJIF Impact Factor 3.628

Research Article

ISSN 2394-3211

EJPMR

EUROPEAN JOURNAL OF PHARMACEUTICAL

AND MEDICAL RESEARCH www.ejpmr.com

ejpmr, 2016,3(11), 484-497

*Corresponding Author: S. P. Chaudhari

Dr. D.Y. Patil College of Pharmacy, Akurdi, Pune-44.

ABSTRACT

The present study concentrates on the improving bioavailability of Metformin HCl which is a BCS-III drug. An

attempt was made to enhance the intestinal permeability of Metformin by forming solid dispersions using β-CD, Pluronic F127 and Gelucire 50/13. The optimize solid dispersion along with dependent and independent variables

like Gelucire 50/13, Pluronic F 127, which are waxy non –ionic surfactants as lubricant as well as absorption

enhancer with soluble and insoluble diluents were used to formulate the formulation using design expert 9. Twenty

three formulations of immediate release tablet were prepared by using steam granulation method. All the

formulation was evaluated for Content uniformity, Permeability coefficient, Hardness, Friability and Disintegration

Time. Of which F8 formulation was optimized as it shows high permeability than other formulations. This F8

fomulation on comparison with Marketed formulation showed more in-vivo bioavailabilty. Thus from this study it

can be concluded that use of waxy non –ionic surfactants along with β-cyclodextrin played a significant role in

improving the permeability and thus bioavailability of Metformin Hydrochloride.

KEYWORDS: Metformin Hydrochloride, Permeability Coefficient, immediate release, isolated goat intestine.

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toxicity. Acarbose causes gastrointestinal upset diarrhea

and flatulence of Sglt clinical trial recently failed due to

safety concerns. Incretin based drugs causes

gastrointestinal problems such as sour stomach, belching,

nausea, vomiting, indigestion and diarrhea. Insulin

cannot match the natural precise timing and dosing of insulin secretion from the pancreas in response to

hyperglycemia, resulting in severe complications.

Metformin having multiple uses, with few side effects.

Accordingly, the drug's numerous side benefits

associated with the treatment and prevention of diabetes,

as well as other disorders, appear to outweigh its limited

side effects.[5]

All the BCS Class-III drugs show high solubility and low

permeability. Due to low permeability they have less

intestinal absorption. The present study concentrates on the improving bioavailability of Metformin HCl which is

a BCS-III drug.

MATERIALS AND METHOD

Metformin Hydrochloride was obtained as gift sample

from Alkem Laboratories, India, β-cyclodextrin was

obtained as gift sample from Emcure Pharmaceuticals

Pvt. Ltd., Pune while Gelucire 50/13 and Pluronic F

127was obtained from Gattifosse India Pvt. Ltd,

Mumbai. Microcrystalline cellulose and Spray Dried

Lactose was purchased from Research-Lab Fine Chem, Mumbai.

Preparation of Solid Dispersion[6]

Metformin HCl with β-cd, Gelucire50/13 and Pluronic F-

127 in different ratios were prepared by the solvent

method. The drug and carriers were dissolved in ethanol

(10% w/v) and the solvent was subsequently evaporated

at room temperature while stirring. The products were

then stored in an oven at 40°C for 24 h to ensure

complete ethanol evaporation, followed by pulverization

with a mortar and pestle. These Solid dispersions were

used for further study of permeability enhancement.

Evaluation of Permeability with diffusion studies

using goat intestinal membrane The prepared solid dispersions was studied for

permeability by using Franz- diffusion cell technique.

The fresh goat –Intestine was taken as a membrane and

0.1 NHCl was used as diffusion Medium. The

permeability coefficient was determined for further

selection of proportion of permeability enhancers for

final tablet formulations.[7]

Permeability coefficient =kp=[Q/(A*t*(Co-Ci))]

where Q = the quantity of compound transported through

the membrane in time t (min),

Co and Ci = the concentrations of the compound on the outer side (donor side) and the inner side (receptor side)

of the membrane respectively.

A =the area of exposed membrane in cm2.

Usually Co can be simplified as the donor concentration

and Ci as 0.

The units of Kp are cm/min or cm/hr.

Formulation of granules by steam granulation

method

All the ingredients were accurate weighed and was sifted

through sieve and mixed in blender keeping 12±1 rpm for 10 minutes. The dry mix was wetted using steam

from steam generator. The damp mass was passed

through 24# sieve.[8, 9] All prepared granules were dried

at room temperature. The dried granule was sifted

through 20# sieve. and then it was compressed using

rotary tablet press.[10,11,12] Optimization was done using

design expert software9.0. The Box-Behenken design

gave 23 runs for the formulation (table 1 and 2).

Table 1: Variables of the formulation

Independent variables Level

-1 +1

Conc. of Absorbtion enhancer (X1) 10.00 15.00

Conc.of Diluent (X2) 35.00 50.00

Type of absorption enhancer (X3) Pluronic F 127 Gelucire50/13

Type of Diluents(X4) MCC Spray dried lactose

Dependant variables

Y1 Content unifrmity

Y2 Permeability coefficient

Y3 Hardness

Y4 Friability

Y5 Disintegration time in minutes

*Solid dispersion of Metformin equivalent to 250mg was used in all formulations.

Table 2: Formulation table of Immediate release tablet as Design of expert

Run A:Conc.of Absorbtion

enhancer (mg)

B:Conc.of

Diluents(mg)

C:Type of absorption

enhancer D:Type of diluent

1 14.593 43.463 Pluronic 127 MCC

2 15.000 36.827 Gelucire MCC

3 11.921 50.000 Pluronic 127 Spray dried lactose


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4 12 49.85 Gelucire Spray dried lactose

5 15 41 Gelucire Spray dried lactose


7 15.000 36.827 Gelucire MCC

8 15.000 37.818 Gelucire MCC

9 15 35 Pluronic 127 MCC

10 12.9762 43.9456 Pluronic 127 MCC

11 12.9747 35.075 Gelucire MCC


13 10.425 36.275 Gelucire MCC


15 15 50 Pluronic 127 Spray dried lactose


17 12.1 41.075 Gelucire Spray dried lactose

18 14.55 48.8 Gelucire Spray dried lactose

19 12.9747 35.075 Gelucire MCC



22 12.9762 43.9456 Pluronic 127 MCC

23 10 35 Gelucire Spray dried lactose

EVALUATION OF IMMEDIATE RELEASE

TABLETS

Pre-compression Parameters

The prepared granules were evaluated for Bulk density,

tapped density cars index, Hausners ratio, angle of repose.[13,14]

Post –Compression Parameters

The finished tablets were tested for Hardness, Friability,

Disintegration time, Drug content, Permeability

coefficient as per following standard procedure.

Hardness The hardness was determined for 10 tablets of each batch

by using Monsanto tablet hardness tester. The average

was determined.

Friability

Twenty tablets were weighed and placed in the

Electrolab friabilator and the apparatus was rotated at 25

rpm for 4 minutes. After revolutions the tablets were

deducted and weighed again. The percentage friability

was measured using the formula,

% F = {1-(Wt/W)} ×100

Where, % F = friability in percentage

W = Initial weight of the tablet

Wt = weight of tablets after revolution.

Disintegration time

Disintegration is evaluated to ensure that the drug

substance is fully available for dissolution and absorption

from the gastrointestinal tract. The disintegration test

was done on 6 units using the apparatus described in

United States pharmacopoeia method.

Drug content The assay of Metformin HCl in tablets was estimated by

UV method.

Sample preparation- 20 Tablets were weighed and finely

powdered. About 10 mg equivalent of Metformin HCl

was transferred in to 100 ml volumetric flask added with

about 10 ml of methanol and sonicated it for 10 min and

the volume was made up with distilled water. The solution was filtered and diluted1 ml of filtrate to 100 ml

with distilled water and subjected for UV detection.

Absorbance of sample and standard preparation were

measured at 233±2 nm using distilled water as blank.

In-vitro permeability studies[15]

Isolation of goat intestine The Krebs-Ringer solution was prepared by adding 6.3g

NaCl, 0.35g KCl, 0.14 g CaCl2, 0.16 g KH2PO4, 0.15 g

MgSO4.7H2O, 2.1g NaHCO3 and 5g glucose to one liter

of distilled water. The fresh goat intestinal lumen was

carefully cleared from mucus by rinsing with a pH 7.4 buffer solutions. An intestinal segment of the first 6-cm

length was removed and transferred to oxygenated

Krebs-Ringer solution. The proximal extremity of the

intestine was turned back and ligated on a glass rod to

form an everted bag.

Design of simultaneous Dissolution–Absorption

System Using goat Intestine Segment The in vitro continuous dissolution–absorption system

design is illustrated in Figure.no.1. The system consisted

of USP dissolution Apparatus 1 and a side-by-side perfusion apparatus holding isolated everted intestine

segment.

The dissolution medium used was 9000 mL of 0.1 N HCl

maintained at 37 ± 0.5°C. The perfusion apparatus

consisted of two glass tubes, A and B, connected

together. Tube B had a bent cannula at its lower end and

tube A, a straight cannula at its lower end. The distance

between the two cannula was kept constant. The fresh

isolated intestinal segment was fixed between the ends of


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tubes A and B as shown in the Figure no.1. The ends of

the intestine were tied in position with a thread. The

apparatus was immersed completely into the dissolution

vessel. The total volume of the absorption compartment

(tube A and tube B of perfusion apparatus) was 35 ml of

Krebs–Ringer solution. The drug diffused from dissolution compartment to the absorption compartment.

The tablet was transferred to the dissolution basket of the

designed system. The tablet was rotated at 75 rpm speed.

The drug was transported from dissolution compartment

to absorption compartment. The transported drug from

the absorption compartment was sampled with

replacement (Krebs–Ringer solution) at 10min., 20min,

30min,1 hr.,1.5hrs, 2hrs and analyzed

spectrophotometrically for transported Metformin HCl at

232 nm. The same procedure was preceeded for all the

formulation also.

Formula for calculation of permeability coefficient P (cm/sec)= Dq/ dt ×1 /60×A×C0

Dq/dt=Amount of drug traversing the tissue in time t

A=Exposed area of tissue

C0=Initial concentration of drug in donar compartment

Figure 1: Continuous Dissolution–Absorption System

In -vivo bioavailability study[22,23]

The pharmacokinetic interaction of optimized

Formulated Metformin HCl tablet with Marketed Tablet

was studied in normal Wistar Albino rats.

Blood samples were analyzed using a validated HPLC

method to generate the pharmacokinetic profile of

metformin HCl. The C max and T max, AUC and t ½

were calculated from the concentration–time data.

Selection of Animals Wistar Albino rats (200–280 g) of either sex were

acclimatized in the animal room at least 1 day prior to

the commencement of the study and were maintained on

standard food and water ad libitum. The study protocols

were prepared according to the guidelines specified by

the local animal ethics committee.

Preparation of the formulation The rat dose of metformin was calculated from the

standard clinical human dose on the basis of surface area

[mice dose ={(human dose/average body weight}×7}].

The Formulated tablet and Standard Marketed tablet was

weighed accurately equivalent to the 2.5 mg of

metformin. HCl. Then it was dissolved in 2.5 ml triple-

distilled water to obtain a strength of 1 mg/ml.

Oral administration

Eighteen rats were divided in three groups of 6 each. The overnight fasted rats received a single 10 mg/kg oral

dose of metformin HCl using a rat feeding needle and

syringe. The blood samples were withdrawn from each

rat, through periorbital, puncture (~1.2 ml). Blood

samples from the dosed rats were collected at 0.25, 0.5,

0.75, 1, 1.5, 2, 3, 4 h using a heparinized syringe. It is

collected in Standard EDTA Tube.

Analytical procedures

The HPLC method was developed and validated to

determine the concentration of metformin HCl in rat

plasma. Each 1.2 ml of blood sample was centrifuged for 30 minutes and plasma was separated for further

analysis.

Then 100 μl of plasma was mixed with 200 μl of

acetonitrile and then vortexed for 1 min. Further, these

samples were allowed to stand for 30 min and then

centrifugated at 1000 rpm for 10 min. 20 μL supernatant

was injected onto the HPLC system. The HPLC system


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was equipped with double reciprocating pump,

Rheodyne injector fitted with a fixed 20μL loop, UV-VIS

multiple wavelength detector set at 233 and Ezchrome.

software on personal computer. Separation was obtained

on a C-18 column. Maintaining optimized conditions as

below: Flow rate: 1.0 ml per min, Detector wave length: 233 nm, Column temperature: 25°C, Injection volume:

20 µl, Run time: 10.0 min, Diluent: Water, Mobile

phase: 30:70:1%:1% (water: acetonitrile:

orthophosphoric acid: formic acid) The retention time

was found to be 3.24min.

RESULTS AND DISCUSSION

Preformulation studies were performed on metformin

and the wavelength of maximum absorbance (λ max) of

drug was found to be 232 nm The absorbance reading

with standard solution containing 1-10g/ml showed Slope of 0.0959, intercept of 0.027 and regression

coefficient value of 0.9955 indicate the linear

relationship between absorbance and concentrationand

equation obeyed linearity in range of 2-10 g /ml. Solubility of the Metformin HCl indicated that the drug

was freely soluble in water, slightly soluble in alcohol

and practically insoluble in acetone and in methylene

chloride. The melting point was found to 2340C.

Preliminary studies for increasing permeability of

drug

Metformin being a class III drug has poor permeability,

hence an attempt was made to increase the permeability

by forming solid dispersions using β-CD and Pluronic

F127. In this work, goat intestinal segment was used as a

model for permeability studies. Drug permeation study is

carried out by Franz –diffusion cell method. The

permeability of Metformin hydrochloride across the

excised intestinal segment is shown in table 3.

Table 3: Permeability coefficient of Metformin HCl

Sr.No. Type of Dispersing agent Conc.of Dispersing

Agent: Drug ratio

Permeability

coefficient (cm/sec)

Plain metformin 7.3904 x 10 -5

1 β-cyclodextrin 1:1 15.3598 x10-5

2 β-cyclodextrin 1:2 13.4938x10-5

3 β-cyclodextrin 1:3 9.8626 x10-5

4 β-cyclodextrin 1:4 8.2526 x 10-5

5 β-cyclodextrin 1:5 7.5902 x10 -5

6 Poloxamer-407 1:1 11.2356 x10-5

7 Poloxamer-407 1:2 9.5623 x10-5

8 Poloxamer-407 1:3 8.1235x10-5

9 Poloxamer-407 1:4 4.5623x10-5

10 Poloxamer-407 1:5 3.6456 x10-5

In the β-cd inclusion complex of drug in solution only

free form of the drug, which is in equilibrium, is capable

of penetrating the lipophilic barriers and thus entering

systematic circulation. CDs enhance the mucosal drug

permeability mainly by increasing the free drug

availability at the absorptive surface.[16]

Poloxamers are triblock copolymers of

poly(oxyethylene)–poly(oxypropylene)-poly(oxyethylene). It consisting of 70%w/w

polyoxyethelyne units, is a low toxicity excipient

approved by FDA for different types of preparation.

They are extensively used as solubilizers, wetting agents

and surface adsorption excipients.[17] Poloxamer 407

(Pluronic®F-127 [PL]) is successfully employed as a

solid dispersion carrier in form of matrix dispersion used

as bioavailability enhancer.[18]

The results clearly indicated solid dispersion increase the

permeability of drug compared to plain drug. From

results it is clear that β-cyclodextrin in 1:1 ratio shows more permeability than poloxamer and other ratios.

Hence B-CD in 1: 1 ratio was optimized from

preliminary work for further study.

Evaluation of Prepared granules

Table 4: Results for prepared granules

Formulation

No.

Angle of

repose

Bulk density

(gm/cc)

Tap

density(gm/cc) Hausner ratio Carr’s index

1 31.2 ±1.11 0.89 ±0.02 0.95 ±0.02 1.06 ±0.06 6.31 ±1.23

2 35.2 ±1.42 0.83±0.01 0.93 ±0.01 1.12 ±0.08 10.49±1.46

3 28 ±1.02 0.82 ±0.02 0.90 ±0.02 1.09 ±0.05 8.48 ±2.23

4 38.6 ±0.75 0.82±0.01 0.89 ±0.01 1.09 ±0.01 7.58 ±1.89

5 39.1 ±0.95 0.85 ±0.02 0.97 ±0.02 1.13 ±0.02 13.37 ±1.23

6 29.1 ±1.23 0.75 ±0.01 0.88 ±0.01 1.17 ±0.06 14.77±1.56

7 35.2 ±2.06 0.83 ±0.01 0.93 ±0.02 1.12 ±0.05 10.49±1.21

8 30 ±2.89 0.83 ±0.02 0.89 ±0.01 1.07±0.08 6.74 ±1.36


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9 28.6 ±1.78 0.84 ±0.02 0.92 ±0.02 1.09 ±0.06 8.69 ±1.21

10 25 ±1.23 0.86 ±0.01 0.95 ±0.01 1.10 ±0.03 9.47 ±1.56

11 25.6 ±2.05 0.87 ±0.01 0.93 ±0.02 1.06 ±0.02 6.45 ±1.48

12 28.2 ±1.43 0.88 ±0.02 0.94 ±0.03 1.06 ±0.04 6.38 ±1.25

13 30.6 ±1.11 0.85 ±0.01 0.96 ±0.02 1.129 ±0.05 6.25 ±1.41

14 28.2 ±2.13 0.88 ±0.03 0.94 ±0.01 1.06 ±0.06 6.38±1.13

15 25 ±2.42 0.83 ±0.01 0.93 ±0.03 1.12 ±0.07 10.75±1.07

16 25.6±1.56 0.84 ±0.03 0.91 ±0.02 1.09 ±0.09 7.69 ±1.13

17 28.2±1.86 0.88 ±0.01 0.93 ±0.02 1.05 ±0.01 5.37±1.15

18 29.2 ±1.23 0.89 ±0.02 0.94 ±0.02 1.05 ±0.05 5.31 ±1.01

19 25.6 ±2.46 0.87 ±0.01 0.93 ±0.01 1.06 ±0.08 6.45±1.85

20 29.8 ±2.13 0.75 ±0.01 0.88 ±0.01 1.17 ±0.06 14.77±1.56

21 28 ±1.89 0.82 ±0.01 0.90 ±0.03 1.09±0.05 8.48 ±1.21

22 25 ±1.14 0.86 ±0.02 0.95 ±0.01 1.10 ±0.03 9.47 ±1.21

23 31.2 ±1.26 0.84 ±0.02 0.92 ±0.01 1.09 ±0.08 8.692±1.45

Angle of repose

Rougher and more will be irregular surface of the

particles; higher will be the angle of repose. Angle of

repose of all batches laid between 25.00 ± 0.02 to 39.1±

0.02 which means all batches had good or excellent flow property according to standard.

Bulk density

In all batches F6 had lowest bulk density while F20 had

highest bulk density.

Tapped density In all batches F6 had lowest tapped density while F5 had

highest tap density.

Hausner ratio

The batches exhibited 1.05 ± 0.01 to 1.13± 0.02. Hausner ratios which was within acceptable range according to

standard.

Carr’s index The batches exhibited 5.31± 1.01 to 14.77 ± 1.56 Carr’s

index which was within acceptable range according to

standard.

The granules of 23 formulations were prepared by Steam

granulation method. These Granules were evaluated for

precompression parameters. This method is having

advantage like higher binder distribution uniformity and

diffusion rate into powders, more spherical granule with

large surface area were formed thereby increases

dissolution rate of the drug from granules, favorable

thermal balance results in rapid drying, time efficient as

processing time was shorter and environment friendly as

no organic solvent is used.[19]

Table 5: Results for immediate release tablet

Formulation

No

Content uniformity Permeability

coefficient Hardness Friability Time

% Cm/ sec kg/cm3 % Min.

F1 99.121±0.121 0.001328±0.0012 8.085±0.12 0.711±0.08 5.529±0.01

F2 99.239±0.231 0.001880±0.0016 8.269±0.25 0.00.68±0.03 5.615±0.02

F3 99.138±0.051 0.001243±0.0014 7.511±0.26 0.0066±0.03 5.765±0.03

F4 99.295±0.066 0.001268±0.0014 7.878±0.16 0.0056±0.04 5.786±0.05

F5 99.226±0.071 0.001303±0.0005 7.559±0.18 0.0062±0.05 5.794±0.06

F6 99.136±0.047 0.001194±0.0016 7.504±0.17 0.0071±0.05 5.672±0.01

F7 99.239±0.023 0.001861±0.0018 8.269±0.14 0.0073±0.08 5.615±0.02

F8 99.246±0.045 0.001900±0.0017 8.524±0.04 0.0074±0.02 5.621±0.04

F9 99.121±0.012 0.001388±0.0015 8.085±0.12 0.0072±0.04 5.529±0.05

F10 99.121±0.056 0.001398±0.0017 8.085±0.16 0.0061±0.04 5.532±0.04

F11 99.217±0.087 0.001823±0.0015 8.494±0.15 0.0074±0.06 5.656±0.07

F12 99.138±0.054 0.001256±0.0012 7.513±0.23 0.0062±0.09 5.762±0.05

F13 99.224±0.058 0.001876±0.0017 8.303±0.12 0.0072±0.08 5.661±0.04

F14 99.138±0.047 0.001129±0.0016 7.513±0.23 0.0059±0.07 5.762±0.04

F15 99.137±0.045 0.001134±0.0014 7.51±0.12 0.0061±0.02 5.766±0.02

F16 99.121±0.026 0.001289±0.0018 8.085±0.14 0.0072±0.02 5.53±0.03

F17 99.215±0.09 0.001438±0.0017 7.878±0.14 0.0064±0.03 5.851±0.04

F18 99.232±0.071 0.001402±0.0018 7.587±0.17 0.0063±0.04 5.869±0.05

F19 99.217±0.0251 0.001627±0.0012 8.494±0.12 0.0071±0.05 5.656±0.07


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F20 99.121±0.052 0.001399±0.002 8.084±0.14 0.0078±0.05 5.526±0.04

F21 99.138±0.026 0.001143±0.0014 7.511±0.13 0.0062±0.08 5.765±0.04

F22 99.121±0.023 0.001364±0.0014 8.085±0.15 0.0072±0.07 5.532±0.05

F23 99.222±0.025 0.001444±0.0025 7.541±0.15 0.0069±0.04 5.765±0.04

Content uniformity

Content uniformity of all the formulations were in range

of 99.0 - 99.5%. Showing uniformity of mixing and

compression.

Final Equation in Terms of Coded Factors

Content uniformity=+99.17+0.020* X1+0.055*

X2+0.082* X3-4.845E-003* X4

Graphs of content uniformity

A B

C D

Figure 2: 3 D Surface plot of effect of conc.of diluents and absorption enhancer on content uniformity( A-

PluronicF127 in combination with MCC,B- PluronicF127 in combination with Spray dried Lactose, C- Gelucire

50/13 in combination with Spray dried lactose and D- Gelucire 50/13 in combination with MCC)


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From the above fig no.2 it was cleared that with increase

in conc. of diluent and conc. of absorption enhancer drug

content increases. As such there is negligible difference

in drug content of tablet containing gelucire 50/13 and

pluronic F 127. But as results show that tablets

containing combination of Gelucire 50/13 with MCC and Spray dried lactose showing good content of uniformity

when Compared with tablets containing Pluronic F 127

with the combination of MCC and Spray dried lactose

showing less content of uniformity.

Permeability coefficient

Permeability coefficient of all the formulations were in

range of 0.11-0.19 showing remarkable increase in the

permeability coefficient.

Final Equation in Terms of Coded Factors Permeability through goat intestine= +1.510E-003-4.878E-005* X1+1.975E-005* X2+1.440E-004* X3-

1.791E-004* X4.

From the graph below in Fig.no.3 it was cleared that the

tablets containing gelucire 50/13 showed more

permaeability than tablets containing Pluronic F127.

When the concentration of Gelucire 50/13 was increased

from 5% to 7.5% the Permeability coefficient was

increased. The tablet containing 7.5% concentration of

Gelucire 50/13 showed more Permeability coefficient

than Pluronic F127. Concentration of Absorption

enhancer affect on Permeability coefficient of drug.

Concentration of Gelucire 50/13 has positive effect

might be due to surfactant carrier family that has been described as a being penetration enhancer, a

characteristic attributed to its surfactant and lipid nature.

Lipid surfactants such as Gelucire reportedly cause cell

membrane polar defects, thus changing their physical

properties and making them more permeable than

Poloxamer 407, which is a polymeric surfactant that has

been described as a permeability enhancer. Poloxamer

407 in SDs did not improve the bioavailability, most

likely because ofnthe low intrinsic ability of Poloxamer

407 to favor cell entry.[20,21,22] Gelucire enhances

biological membrane penetration and could thus increase

the bioavailability of drugs.[23] The another reason might be Pluronic F127 is having viscosity around 5800 mPas

while pure Gelucire 50/13exhibited a viscosity of

50mPas. The Pluronic F127 is having a property of

forming gel in the aqueous media. This might be causing

the effect on less drug release and thus less content

uniformity of tablets containing Pluronic F127.[24]

A B


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C D

Figure 3: 3D Surface plot of effect of conc.of diluents and absorption enhancer on Permeability (A-

PluronicF127 in combination with MCC,B- PluronicF127 in combination with Spray dried

Lactose, C- Gelucire 50/13 in combination with Spray dried lactose and D- Gelucire 50/13 in

combination with MCC) Hardness

Hardness of all the formulations is In the range of 7.04-8.72 kg/cm 3

Final Equation in Terms of Coded Factors

Hardness=+7.82+0.097* X1+0.26* X2+0.20* X3-0.35* X4.

A B


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C D

Figure 4: 3 D Surface plot of effect of conc.of diluents and absorption enhancer on Hardness ( A- PluronicF127

in combination with MCC,B- PluronicF127 in combination with Spray dried Lactose,C- Gelucire 50/13 in

combination with Spray dried lactose and D- Gelucire 50/13 in combination with MCC)

From the above fig.4 it was cleared that addition of MCC

as a diluent in Tablets containing Pluronic F127 as well

as Gelucire 50/13 causes increased Hardness. While

addition of spray dried lactose as diluents causes a slight decrease in hardness.

MCC has more free hydroxyl group and thus the

interaction forces in a contact point may be stronger

because of stronger hydrogen bond of hydroxyl groups,

which can cause increase in hardness. This interlocking

phenomenon can have effect of increasing hardness than

that of Spray dried lactose.[25]

Friability The friability of all the formulations is in the range of

0.0054-0.0078%.

Final Equation in Terms of Actual Factors

Friability=+6.566E-003+1.054E-004* X1+6.960E-004*

X2+8.466E-005* X3-4.273E-004* X4.

A B


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C D

Figure 5: 3 D Surface plot of effect of conc.of diluents and absorption enhancer on Friability( A- PluronicF127

in combination with MCC, B- Gelucire 50/13 in combination with MCC,C- Gelucire 50/13 in combination with

Spray dried lactose and D- PluronicF127 in combination with Spray dried Lactose)

From the above fig.no.5 it is cleared that the tablets

containing spray dried lactose shows less friability than

the tablets containing MCC as diluents. The friability is

within the limits of all the formulations that is < 1%.

As spray dried lactose and Microcrystalline cellulose

both enhances rheological properties of wetted mass

resulting in good sphericity and low friability, high

density. Both show friability less than 1%. It might be

during steam granulation slight melting of spray dried lactose might have taken place which increase density of

spray dried lactose than MCC. Hence this slight

difference in friability might be the reason between

friability of tablets containing MCC and spray dried

lactose.[26]

Disintegration

Disintegration time for all the formulations is in the

range of 5.46-6.00 minutes.

Final Equation in Terms of Coded Factors Disintegration =+5.78-0.11* X1+0.046* X2+0.072* X3+0.091* X4.

A B


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C D

Figure 6: 3 D Surface plot of effect of conc.of diluents and absorption enhancer on Disintegration time( A-

PluronicF127 in combination with MCC, B- Gelucire 50/13 in combination with MCC,C- Gelucire 50/13 in

combination with Spray dried lactose and D- PluronicF127 in combination with Spray dried Lactose)

From the above figure no. 6 it was cleared that when the

tablets containing the pluronic F 127 and MCC get

disintegrated rapidly than other tablets. While the tablets

containing gelucire50/13 and Spray dried lactose get disintegrated slowly.

Microcrystalline cellulose is partially depolymerised

cellulose prepared from alpha cellulose. When

compressed, the MCC particles are deformed plastically

due to the presence of slip planes & dislocation. A strong

compact is formed due to the extremely large number of

clean surfaces brought in contact during plastic

deformation & the strength of hydrogen bonds formed.

The mechanism involves is interlocking. MCC has a very

high intraparticle porosity with approximately 90–95%

of the surface area being internal. Therefore surface area is not directly influenced by the nominal particle size.

This high porosity promotes swelling and disintegration

of MCC tablets, which is attributed to the penetration of

water into the hydrophilic tablet matrix by means of

capillary action of the pores and by a subsequent

disruption of the hydrogen bonds MCC allowing water to

enter the tablet matrix by means of capillary pores,

which break the hydrogen bonding between adjacent

bundles of cellulose microcrystals and exhibit very good

disintegrant property. The particle size of MCC is small.

The decrease in particle size increases binding strength

and decreases disintegration time. MCC is found in the

concentration of 10-25% as a filler, binder, disintegrant.

MCC is useful as a disintegrant when used in proportion

of at least 5-15%.[27]

While the Tablets containing spray dried lactose

exhibited increase in crushing strength with decrease in

the particle size. The disintegration time of spray-dried

lactose tablets was essentially independent of

compaction force. Due to compaction pressure, tablets

containing spray dried lactose disintegrated before gel or

precipitate could block the pores. This gelling and

precipitation dominated the disintegration time. Spray-

dried lactose exhibited strong increase in disintegration

time.[28]

In-vivo bioavailability study

HPLC method showed the good peak with good

symmetry. Hence this method was finalized for the

development of metformin.

The Albino rats treated with both Optimized Formulated

Metformin Tablet and Marketed Tablet. Glycomet- 250

mg (USV LIMITED) was used as the marketed

formulation. The result showed that, the C max of

Formulated tablet was found to be higher than that of

marketed formulation.

Table 5: Pharmacokinetic parameters for In-vivo bioavailability study.

Parameters Formulated tablet Marketed tablet

C max (ng) 70.74 16.78

T max (min) 9.12 11.24

AUC( 0-4hr) (ng/min/ml) 2.1676 1.468

t ½ (min.) 12.38 13.58


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CONCLUSION

The in –vivo evaluation of optimized formulation with

marketed formulation showed improvement in

Bioavailability than Marketed formulation. Hence it can

be concluded that use of waxy non –ionic surfactants

played a significant role in improving the permeability and thus bioavailability of Metformin Hydrochloride.

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STUDY OF FORMULATION VARIABLES ON BIOAVAILABILITY …